Protein ubiquitination has emerged as a crucial mechanism for controlling development and function of neuronal circuits, and its defective regulation has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. However, very little is presently known about the molecular machinery that controls protein ubiquitination in neurons. In the ubiquitin-proteasome pathway, substrate proteins are marked for degradation in the proteasome by covalent linkage to ubiquitin, a 76-amino acid polypeptide. The ubiquitination process involves a highly specific enzyme cascade in which ubiquitin is first activated by an E1 ubiquitin-activating enzyme, then transferred to an E2 ubiquitin-conjugating enzyme, and finally ligated to the substrate by an E3 ubiquitin-protein ligase. Of these enzymes, E3 ligase is the most important player because it determines the specificity of ubiquitin-mediated protein degradation. The importance of E3 ligases in neurodegenerative disorders is highlighted by recent findings that mutations in the E3 ligase parkin are responsible for a familial form of Parkinson's disease. In a search for neuronal proteins that regulate the neurotransmitter release machinery component SNAP-25, the applicant has discovered a novel protein, called Spring. Spring is a neuron-specific member of the RING-B-box-coiled-coil (RBCC) protein family. The importance of the RBCC family is underscored by the identification of the mutations in several RBCC proteins as the causes for a number of human diseases, including Opitz syndrome, Mulibrey nanism, and familial Mediterranean fever. In this project, the applicant will use a combination of biochemical, proteomic, molecular biological, and cell biological approaches to test the hypothesis that Spring functions as a novel E3 ubiquitin-protein ligase to regulate the turnover of the neurotransmitter release machinery. In addition, this project will characterize neuronal distribution and synaptic localization of Spring, and explore the possible involvement of this novel protein in Alzheimer's disease and Parkinson's disease. Successful completion of proposed studies will yield novel insights into the molecular mechanisms that control neuronal protein ubiquitination and neurotransmitter release, and provide fundamental information towards our ultimate goal of understanding and treating numerous neurological diseases and psychiatric disorders.